This is a continuing proposal. The primary objective of this proposal is to understand the basic mechanistic and structural properties, which account for fidelity, or the lack thereof, in DNA polymerases. For the next funding period, two DNA polymerases with contrasting properties will be studied: a high-fidelity enzyme, rat Pol beta, and a low-fidelity enzyme, African Swine Fever virus (ASFV) Pol X. The Specific Aim 1 is to elucidate the mechanism of fidelity of Pol beta by determining the microscopic rate constants for all steps in the reaction pathway for correct and mismatched base pair formation. This will allow a precise determination of the contribution each step makes to catalysis and fidelity. The Specific Aim 2 is to understand the mechanism of low fidelity of Pol X by detailed mechanistic comparison between Pol beta and Pol X, which will provide a unique opportunity to examine the differences between high-fidelity and low-fidelity DNA synthesis. Studies of site-directed mutants will also be employed to examine how the two enzymes utilize specific residues to achieve high fidelity and low fidelity. The Specific Aim 3 is to determine the structures of Pol X bound to DNA and dNTP by NMR. The structures of the complexes with mismatched dNTP would represent a substantial advance in polymerase structural work. In addition, there are two secondary objectives. One is to identify and characterize chimeric DNA polymerases constructed by gene shuffling, with the goal of identifying chimeric polymerases which display enhanced activity and/or novel specificity (specific Aim 4). The other is to characterize the ASFV DNA ligase, with the aim of testing our hypothesis that ASFV utilizes a low-fidelity polymerase and a low-fidelity DNA ligase to increase randomization of the viral genome (Specific Aim 5). Each of the studies in this proposal represents a continuation or extension of our ongoing studies in this area.